Transcriptome analysis based on RNA-seq of common innate immune responses of flounder cells to IHNV, VHSV, and HIRRV.

Kwang Il Kim,Eun Young Min,Sung-Hee Jung,Jeong Woo Park,Unn Hwa Lee,Miyoung Cho,Maria Del Mar Ortega-Villaizan

doi:10.1371/journal.pone.0239925

Abstract

Viral hemorrhagic septicemia virus (VHSV) and hirame rhabdovirus (HIRRV) belong to the genus Novirhabdovirus and are the causative agents of a serious disease in cultured flounder. However, infectious hematopoietic necrosis virus (IHNV), a prototype of the genus Novirhabdovirus, does not cause disease in flounder. To determine whether IHNV growth is restricted in flounder cells, we compared the growth of IHNV with that of VHSV and HIRRV in hirame natural embryo (HINAE) cells infected with novirhabdoviruses at 1 multiplicity of infection. Unexpectedly, we found that IHNV grew as well as VHSV and HIRRV. For successful growth in host cells, viruses modulate innate immune responses exerted by virus-infected cells. Our results suggest that IHNV, like VHSV and HIRRV, has evolved the ability to overcome the innate immune response of flounder cells. To determine the innate immune response genes of virus-infected HINAE cells which are commonly modulated by the three novirhabdoviruses, we infected HINAE cells with novirhabdoviruses at multiplicity of infection (MOI) 1 and performed an RNA sequencing-based transcriptome analysis at 24 h post-infection. We discovered ~12,500 unigenes altered by novirhabdovirus infection and found that many of these were involved in multiple cellular pathways. After novirhabdovirus infection, 170 genes involved in the innate immune response were differentially expressed compared to uninfected cells. Among them, 9 genes changed expression by more than 2-fold and were commonly modulated by all three novirhabdoviruses. Interferon regulatory factor 8 (IRF8), C-X-C motif chemokine receptor 1 (CXCR1), Toll/interleukin-1 receptor domain-containing adapter protein (TIRAP), cholesterol 25-hydroxylase (CH25H), C-X-C motif chemokine ligand 11, duplicate 5 (CXCL11.5), and Toll-like receptor 2 (TLR2) were up-regulated, whereas C-C motif chemokine receptor 6a (CCR6a), interleukin-12a (IL12a), and Toll-like receptor 1 (TLR1) were down-regulated. These genes have been reported to be involved in antiviral responses and, thus, their modulation may be critical for the growth of novirhabdovirus in flounder cells. This is the first report to identify innate immune response genes in flounder that are commonly modulated by IHNV, VHSV, and HIRRV. These data will provide new insights into how novirhabdoviruses survive the innate immune response of flounder cells.

Highlights

Novirhabdovirus belongs to the family Rhabdoviridae and causes disease in cultured fish, resulting in significant economic losses to aquaculture industries
To determine whether infectious hematopoietic necrosis virus (IHNV) grows in flounder cells as efficiently as viral hemorrhagic septicemia virus (VHSV) and hirame rhabdovirus (HIRRV), we compared infectious virus yields of IHNV, VHSV, and HIRRV in a flounder cell line, hirame natural embryo (HINAE), at 20 ̊C
At day 3 and day 5 p.i., even though the titers of the three novirhabdoviruses showed a slight difference, the viral yield of all three significantly increased and the growth curve of IHNV was similar to those of VHSV and HIRRV (Fig 1). These results suggest that IHNV is competent to replicate in flounder cells as VHSV and HIRRV

Summary

Introduction

Novirhabdovirus belongs to the family Rhabdoviridae and causes disease in cultured fish, resulting in significant economic losses to aquaculture industries. In addition to IHNV, viral hemorrhagic septicemia virus (VHSV) and hirame rhabdovirus (HIRRV) are members of the genus Novirhabdovirus. In Asia, VHSV infection was first reported in olive flounder (Paralichthys olivaceus) cultured in Japan in 1996 [17] and caused disease and serious economic problems in olive flounder farming in Japan [18, 19] and Korea [20, 21]. HIRRV was first described in cultured olive flounder in Japan in the early 1980s [22], from where it gradually spread to South Korea, China [23, 24], and Europe [25] and infected a wide range of marine fishes including olive flounder, stone flounder (Kareius bicoloratus), black seabream (Acanthopagrus schlegeli), and spotted sea bass (Lateolabrax maculatus) [26]

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